Considerable effort has been made to develop debridement preparations that are capable of removing devitalized tissue without surgery. Devitalized tissue is formed in all disease processes, which are associated with skin trauma, such as decubitus ulcers, pressure necroses, incisions and burns. Efficient debridement is essential since devitalized tissue is an excellent culture medium for opportunistic infections. Septicemia resulting from infections is the major cause of death for the majority of severely burned patients.
Use of proteolytic enzymes and chemical agents to effect early debridement of devitalized tissue has not led to satisfactory debridement. The chemical agents such as tannic acid, salicylic acid, and pyruvic acid, were found to cause further damage to already injured tissues.
Proteolytic enzymes including papain, pinguinain, trypsin, fibrinolysin, and streptokinase have been described as debriding agents. U.S. Pat. No. 5,505,943 discloses compositions containing a protease produced by microorganisms of the genus Vibrio for treating wounds by hydrolyzing components of necrotic tissue.
However, debridement by purified proteolytic enzymes suffers from various disadvantages as the purified enzymes require numerous applications over a long period of time, show poor efficacy, have toxic side effects and have no selectivity.
Extracts derived from the stem of the pineapple plant (Ananas comosus) have been found to selectively remove devitalized tissue. Such extracts, also named bromelain, contain various proteolytic and hydrolytic enzymes.
U.S. Pat. No. 4,197,291 discloses an enzyme product obtained from bromelain capable of debridement of devitalized tissue from a mammalian host, the enzyme product comprises a water soluble, heat labile protein that is free of caseinolytic activity and has a peak isoelectric point of about 6. The protein comprises at least two subunits, each of which having a molecular weight from about 14.3 to 15 kDa with a characteristic absorption peak in the ultraviolet region of the spectrum at 280 nm. The procedure to prepare such enzyme product as disclosed in U.S. Pat. No. 4,197,291 comprises protein precipitation with acetone, extraction of the precipitate with acetate buffer containing thioglycolic acid, filtration of the solution through a membrane with a molecular weight cut off of about 50 kDa, gel filtration of the filtrate, and isoelectric focusing. The enzyme product thus prepared contains at least two, most likely three, subunits having a molecular weight of 14.3 to 15 kDa. U.S. Pat. No. 4,226,854 discloses a method for debridement of devitalized tissue using the enzyme product disclosed in U.S. Pat. No. 4,197,291.
U.S. Pat. No. 4,329,430 further discloses a proteolytic enzyme mixture derived from bromelain useful for dissecting and digesting devitalized tissue. The proteolytic enzyme mixture which is heat labile and water soluble contains escharase, a hydrolytic enzyme free of caseinolytic activity with an isoelectric point of about 6, which comprises at least two subunits, each of which has a molecular weight from about 14.3 to 15 kDa. All of the components of the proteolytic enzyme mixture have a native molecular weight of from about 30 to 50 kDa as the enzyme mixture is filtered through a membrane having a molecular weight cut off of 50 kDa and concentrated over a membrane having a molecular weight cut off of 30 kDa. The reproducible results obtained with the proteolytic enzyme mixture are purportedly due to the fact that the enzyme mixture does not contain an inhibitor, which was apparently present in the previously published proteolytic enzyme preparations. However, there is no criterion for the detection of the activity of this inhibitor.
U.S. Pat. No. 4,307,081 discloses a method of dissecting and digesting devitalized tissue, which comprises contacting the tissue with the proteolytic enzyme mixture disclosed in U.S. Pat. No. 4,329,430.
The pineapple plant has been the source of various proteolytic enzymes. For example, U.S. Pat. No. 5,106,621 discloses purified cysteine proteinases derived from pineapple plant material having a molecular weight of about 25 kDa and exhibiting activity toward a coumarylamide substrate. Particularly, U.S. Pat. No. 5,106,621 relates to the cysteine proteinases ananain and comosain, which exhibit different physicochemical characteristics distinct from stem bromelain. A purified thiol activated protease having a molecular weight of about 17 kDa to 21 kDa, named α-Bromelain, is disclosed in U.S. Pat. No. 5,387,517, and is shown to have debridement activity. In addition, bromelain contains an acid phosphatase and a peroxidase and may contain amylase and cellulase activity. U.S. Pat. No. 6,335,427 teaches the purification of a 25 kDa protein from bromelain, the protein has been found to have anti-cancer activity.
Perlstein and Kezdy (J. Supramol. Struct. 1: 249-254, 1973) identified seven closely related protease inhibitors, i.e., bromelain inhibitor I-VII, from commercial bromelain acetone powder. The inhibitors were shown to have molecular weights of 5000-6000 Dalton and to contain 50 amino acid residues and five disulfide bonds (Perlstein and Kezdy, ibid). Primary structural analysis of one of the seven inhibitors revealed extensive microheterogeneity (Reddy, M. N. et al. J. Biol. Chem. 250: 1741-1750, 1975).
U.S. Pat. No. 5,830,739 discloses methods for preparing a stable admixture of escharase and other proteolytic enzymes from bromelain, which comprise extracting bromelain with a dilute ascorbic acid solution, followed by precipitating the escharase and other proteolytic enzymes with ammonium sulfate. U.S. Pat. No. 5,830,739 further teaches that the ammonium sulfate precipitate can be washed with distilled water over a 10 kDa ultrafilter. The method for preparing the stable admixture of escharase is found to yield higher amounts of escharase. However, there is no indication that the admixture is devoid of bromelain inhibitors.
Purified distinct enzymes isolated from bromelain were found to be not as efficient in debridement of non-viable tissues as a proteolytic enzyme mixture obtained from bromelain. However, proteolytic enzyme mixture obtained from bromelain, which contains proteins of molecular weights of up to 50 kDa including bromelain inhibitors was found to be not as effective in debridement of non-viable tissues as an enzyme mixture obtained from bromelain containing proteins of molecular weights of 30 to 50 kDa, which presumably was devoid of the inhibitors.
Since such enzyme mixtures are intended for human clinical use, there is an unmet need to obtain a biochemically characterized enzyme mixture from bromelain, which has distinct and reproducible biochemical features, essentially devoid of bromelain inhibitors and containing most of the proteolytic enzymes of bromelain, so that efficient debridement of non-viable tissues is obtained.